CN102199578B

CN102199578B - Mutant enzyme of glutamate dehydrogenase and construction method thereof

Info

Publication number: CN102199578B
Application number: CN 201110059834
Authority: CN
Inventors: 朱国萍; 潘蔚; 宋平; 曹正宇; 王鹏; 王晖; 王文才; 靳明明
Original assignee: Anhui Normal University
Current assignee: Anhui Normal University
Priority date: 2011-03-14
Filing date: 2011-03-14
Publication date: 2013-06-19
Anticipated expiration: 2031-03-14
Also published as: CN102199578A

Abstract

The invention discloses mutant enzyme of glutamate dehydrogenase and a construction method thereof. The mutant enzyme has an amino acid sequence shown as SEQ ID NO: 1. The mutant gene is well expressed in a heterologous host, namely Escherichia coli; and expressed proteins are one kind of dehydrogenases which can catalyze various amino acids. The method mainly comprises the following steps of: (1) obtaining a glutamate dehydrogenase gene gdh and constructing a recombinant plasmid; (2) constructing a mutant strain; (3) constructing an expression strain; and (4) expressing and purifying. Compared with the prior art, the generated engineering bacteria not only can catalyze glutamic acid, but also can catalyze methionine and norleucine.

Description

A kind of mutant enzyme of glutamate dehydrogenase and construction process thereof

Technical field

The present invention relates to utilize mutant enzyme and construction process thereof, belong to especially mutant enzyme and the construction process thereof of glutamate dehydrogenase.

Background technology

Amino acid dehydrogenase family (amino acid dehydrogenases, EC 1.4.1), this family protein utilizes NAD ⁺Perhaps NADP ⁺For cofactor catalytic amino acid oxidase deamination forms corresponding ketone acid.React as follows:

This family comprises glutamate dehydrogenase (Glutamate dehydrogenase, GDH), leucine dehydrogenase (Leucine dehydrogenase), valine dehydrogenase (Valine dehydrogenase) and phenylalanine desaturase (Phenylalanine dehydrogenase).Because these amino acid dehydrogenases can the specific reaction of catalysis, they are often used in diet and medicine is amino acid whose synthetic, can also be used for the clinical detection of amino acid and ammonia.But it is similar different at aspects such as substrate specificity, stability and salt tolerances that some members of this family share sequential structure.

Glutamate dehydrogenase (Glutamate dehydrogenase, GDH) can be with NAD (P) ⁺Reversible reaction for coenzyme catalysis glucose oxidation deamination and reduction ammonia.Extensively being present in the organisms such as bacterium, animal and plant, is the important enzyme that connects carbon and nitrogen metabolism, significant for vital movements such as the balance of intracellular environment oxidation-reduction quality and biological metabolisms.Glutamate dehydrogenase is the key enzyme in the synthetic glutamic acid metabolism process of glutamate producing bacterium, can also be used for enzyme electrodes makes, preparation of medical reagent urea nitrogen test kit etc. in medical diagnosis such as medicine industry, GDH measures and also can be used as conventional liver function test item, is specially adapted to the judgement of chronic hepatitis patients hepar damnification degree, observation of curative effect and state of illness monitoring.In addition it or the candidate new molecule of malaria quick diagnosis.But above-mentioned enzyme can only catalysis L-glutamic acid, can not catalysis other amino acid, purposes has been subject to certain restriction.

Summary of the invention

The 1st technical problem to be solved by this invention is to provide a kind of mutant enzyme of glutamate dehydrogenase that can the catalysis multiple amino acids.

The 2nd technical problem to be solved by this invention is the construction process of said mutation body enzyme.

The present invention obtains gdh gene from Salmonella typhimurium (Salmonella typhimurium), obtained the gene fragment of sudden change by mutant primer, the aminoacid sequence of its coding as shown in sequence table, the mutator gene fragment is connected with the pET28b plasmid, successfully builds recombinant expression plasmid pET28b-KA/LG; This recombinant plasmid transformed competent escherichia coli cell, the genetic engineering bacterium Rosetta (DE3) that structure is expressed for mutant enzyme/pET28b-KA/LG; Obtained to express preferably under 37 ℃ of normal temperature and 0.5mM IPTG condition.

The present invention compared with prior art, the engineering bacteria that generates can not only catalysis L-glutamic acid, and can also catalysis methionine(Met) and nor-leucine.

Description of drawings

Fig. 1 is the pcr amplification figure of gdh gene.

M:Marker III; The pcr amplification product of 1:gdh gene

Fig. 2 is A166G fragment PCR amplification figure

M:Marker III; 1:A166G downstream fragment; 2:A166G upstream fragment

Fig. 3 is that A166G merges pcr amplification figure

M:Marker III; 1:A166G merges the PCR product

Fig. 4 is KA/LG fragment PCR amplification figure

M:Marker III; 1:KA/LG downstream fragment; 2:KA/LG upstream fragment

Fig. 5 is that KA/LG merges pcr amplification figure

M:Marker III; 1:KA/LG merges the PCR product

Embodiment

The present invention is described in detail below in conjunction with embodiment.

Embodiment 1:

1, gdh gene gdh obtains

According to the known gdh gene design primer of Salmonella typhimurium atcc 14028 (Salmonella typhimurium), take the S.typhimurium genomic dna as template, pcr amplification, result only obtain 1 specific fragment (Fig. 1).Gdh amplimer and PCR condition are respectively:

Sense：5′-GAACCACGTCATATGGATCAGACATGTTC-3′；

Anti-sense：5′-ATCCCTCGAGAAAGCTATCTGGCCTGAC-3′；

95 ℃ of denaturation 3min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min 30s circulate 35 times; 72 ℃ are fully extended 10min.

2, recombinant vectors pET28b-gdh builds

The product of amplification and carrier pET28b after NdeI, Xho I double digestion, connect Transformed E .coliDH5 α respectively.Screening positive clone obtains expression plasmid pET28b-gdh.After double digestion is identified, send that the Nanjing spun gold is auspicious to check order, sequence alignment is found in full accord on sequenced genes and Genbank (GeneBank accession NO.NP_460265).

3, the structure of mutants which had

The design mutant primer, carry out pcr amplification take recombinant plasmid pET28b-gdh DNA as template, this fragment is connected with the pET28b carrier, construction recombination plasmid pET28b-A166G, transform intestinal bacteria E.coli DH5 α competent cell, through blue hickie screening, select the white clone and extract plasmid, send spun gold auspicious order-checking in Nanjing to identify.166 mutant primers and PCR condition are respectively:

166 anti-sense:5 '-GATATCCCCCCCAGGCACGTCG-3 ';

166 sense:5 '-GATACCGACGTGCCTGGGGGGGATA-3 ';

Upstream fragment (fragment that contains the gdh initiator codon): 94 ℃ of denaturation 3min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 30s circulate 35 times; 72 ℃ are fully extended 10min,

Downstream fragment (fragment that contains the gdh terminator codon): 94 ℃ of denaturation 3min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min circulate 35 times; 72 ℃ are fully extended 10min.Pcr amplification the results are shown in Figure 2.

The PCR product merges PCR again after gel reclaims, merge the PCR condition to be: 94 ℃ of denaturation 3min; 94 ℃ of 30s, 64 ℃ of 40s, 72 ℃ of 1min 30s circulate 35 times; 72 ℃ are fully extended 10min.The results are shown in Figure 3.

The product of amplification and carrier pET28b after NdeI, Xho I double digestion, connect Transformed E .coliDH5 α respectively.Screening positive clone obtains expression plasmid pET28b-A166G, carries out pcr amplification take recombinant plasmid pET28b-A166G as template, and this fragment is connected with the pET28b carrier, builds pET28b-KA/LG,

92 mutant primers are:

92 anti-sense:5 '-GCATACCGCC AAGATAGGGGCCGATAG-3 ';

92 sense:5 '-TCGGCCCCTAT CTTGGCGGTATGCG-3 '.

The PCR condition is upstream fragment (fragment that contains the gdh initiator codon): 94 ℃ of denaturation 3min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 35s circulate 35 times; 72 ℃ are fully extended 10min, downstream fragment (fragment that contains the gdh terminator codon): 94 ℃ of denaturation 3min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min circulate 35 times; 72 ℃ are fully extended 10min.The fragment PCR detected result is seen Fig. 4.

The PCR product merges PCR again after gel reclaims, merge the PCR condition to be: 94 ℃ of denaturation 3min; 94 ℃ of 30s, 64 ℃ of 40s, 72 ℃ of 1min 30s circulate 35 times; 72 ℃ are fully extended 10min.Result as shown in Figure 5.

Sequence alignment is found to have suddenlyd change successfully, and expression plasmid pET28b-KA/LG is converted into E.coliRosetta (DE3) competent cell, builds recombinant bacterial strain Rosetta (DE3)/pET28b-KA/LG.

4, the expression of two mutant strains and purifying

The picking recombinant bacterium in the test tube that contains 5ml LB liquid nutrient medium, 37 ℃, 225rpm, the enlarged culturing of spending the night; Culture was inoculated in the tap web bottle that contains 50ml LB liquid nutrient medium with 1: 100, and 37 ℃, 225rpm, shaking culture is to OD ₆₀₀Be 0.6-0.8; Add 0.5mM isopropylthiogalactoside (isopropyl-β-D-thiogalactopyranoside, IPTG), 37 ℃ of abduction delivering 10h; 4 ℃, 5,000rpm, centrifugal 5min collects thalline, ultrasonic disruption.Use Co ²⁺The ion affinity column ( Purification Kit) purifying.Protein after purifying identifies through 10%SDS-PAGE, at molecular weight 47kDa place, the very high band of one specificity arranged, and illustrates that the purity of protein after purifying is higher.SDS-PAGE shows that the molecular size range of this protein is about 47kDa, and is consistent with the molecular size range that calculates by aminoacid sequence.

5, the enzyme kinetics parameter detecting of two mutant enzymes (KA/LG) and with the comparison of the parameter of wild-type StGDH

Be 50mM, NADP at Tris-HCl (pH8.0) final concentration ⁺Final concentration is in the fixing reaction system of 250 μ M, changes the final concentration of L-glutamic acid, with Cary 300 Bio UV-Visible spectrophotometers, detects A under 25 ℃ ₃₄₀Variation, parallel 3～4 secondary responses of doing of identical aminoglutaric acid concentration are averaged and are made curve, reciprocal equation formulas two according to Lineweaver-Burk are calculated the right K of each enzyme _mAnd k _catValue.Use the same method and calculate each enzyme to the K of L-Met and L-Nle _mAnd k _catValue.Result shows that this enzyme not only can be take L-glutamic acid as substrate, can also be take methionine(Met) or nor-leucine as substrate.Their K _mValue is respectively 438.76mM, 68.48mM, and 26.81mM, concrete numerical value sees Table 1.

Transformation by several amino-acid residues makes the glutamate dehydrogenase conversion for a kind of amino acid dehydrogenase of novelty, can be take METHIONINE or L-nor-leucine as substrate.This has just illustrated that also these amino acid are important amino acids that glutamate dehydrogenase and substrate Pidolidone are identified mutually.

Not only make us obtain a kind of enzyme of novelty by such transformation, and enlarged its substrate specificity, can utilize simultaneously several different substrates, this has not only increased the diversity of enzyme, and very large value is also arranged on industrial applicability.Transformation by protein engineering changes the substrate specificity of amino acid dehydrogenase, for the production of or detect natural or alpha-non-natural amino acid.

SEQUENCE LISTING

＜110〉Anhui Normal University

＜120〉a kind of mutant enzyme of glutamate dehydrogenase and construction process thereof

<130>1

<160>1

<170>PatentIn version 3.3

<210>1

<211>448

<212>PRT

＜213〉Salmonella typhimurium (Salmonella typhimurium)

<400>1

His Met Asp Gln Thr Cys Ser Leu Glu Ser Phe Leu Asn His Val Gln

1 5 10 15

Lys Arg Asp Pro His Gln Thr Glu Phe Ala Gln Ala Val Arg Glu Val

20 25 30

Met Thr Thr Leu Trp Pro Phe Leu Glu Gln Asn Pro Arg Tyr Arg His

35 40 45

Met Ser Leu Leu Glu Arg Leu Val Glu Pro Glu Arg Val Ile Gln Phe

50 55 60

Arg Val Val Trp Leu Asp Asp Lys Asn Gln Val Gln Val Asn Arg Ala

65 70 75 80

Trp Arg Val Gln Phe Asn Ser Ala Ile Gly Pro Tyr Leu Gly Gly Met

85 90 95

Arg Phe His Pro Ser Val Asn Leu Ser Ile Leu Lys Phe Leu Gly Phe

100 105 110

Glu Gln Thr Phe Lys Asn Ala Leu Thr Thr Leu Pro Met Gly Gly Gly

115 120 125

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Glu Gly Glu Val

130 135 140

Met Arg Phe Cys Gln Ala Leu Met Thr Glu Leu Tyr Arg His Leu Gly

145 150 155 160

Pro Asp Thr Asp Val Pro Gly Gly Asp Ile Gly Val Gly Gly Arg Glu

165 170 175

Val Gly Phe Met Ala Gly Met Met Arg Lys Leu Ser Asn Asn Ser Ser

180 185 190

Cys Val Phe Thr Gly Lys Gly Leu Ser Phe Gly Gly Ser Leu Ile Arg

195 200 205

Pro Glu Ala Thr Gly Tyr Gly Leu Val Tyr Phe Thr Glu Ala Met Leu

210 215 220

Lys Arg His Gly Leu Gly Phe Glu Gly Met Arg Val Ala Val Ser Gly

225 230 235 240

Ser Gly Asn Val Ala Gln Tyr Ala Ile Glu Lys Ala Met Ala Phe Gly

245 250 255

Ala Arg Val Val Thr Ala Ser Asp Ser Ser Gly Thr Val Val Asp Glu

260 265 270

Ser Gly Phe Thr Pro Glu Lys Leu Ala Arg Leu Cys Glu Ile Lys Ala

275 280 285

Ser Arg Asp Gly Arg Val Ala Asp Tyr Ala Arg Glu Phe Gly Leu Thr

290 295 300

Tyr Leu Glu Gly Gln Gln Pro Trp Ser Val Pro Val Asp Ile Ala Leu

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Leu Asp Val Asp Ala Ala Arg Val Leu

325 330 335

Ile Ala Asn Gly Val Lys Ala Val Ala Glu Gly Ala Asn Met Pro Thr

340 345 350

Thr Ile Glu Ala Thr Asp Leu Phe Leu Glu Ala Gly Val Leu Phe Ala

355 360 365

Pro Gly Lys Ala Ala Asn Ala Gly Gly Val Ala Thr Ser Gly Leu Glu

370 375 380

Met Ala Gln Asn Ala Ala Arg Leu Ser Trp Lys Ala Glu Lys Val Asp

385 390 395 400

Ala Arg Leu His His Ile Met Leu Asp Ile His His Ala Cys Val Glu

405 410 415

Tyr Gly Gly Asp Asn Lys His Thr Asn Tyr Val Gln Gly Ala Asn Ile

420 425 430

Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Ile

435 440 445

Claims

1. the mutant enzyme of a glutamate dehydrogenase, it is characterized in that: it is comprised of the aminoacid sequence shown in SEQ ID NO:1.

2. the gene of the mutant enzyme of the described glutamate dehydrogenase of claim 1 of encoding.